Mouna Stayoussef

Association of single nucleotide polymorphisms in cytotoxic T-lymphocyte antigen 4 and susceptibility to autoimmune type 1 diabetes in Tunisians.

ABSTRACT In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.

Influence of common and specific HLA-DRB1/DQB1 haplotypes on genetic susceptibilities of three distinct Arab populations to type 1 diabetes.

ABSTRACT The contribution of HLA DRB-DQB to type 1 diabetes (T1D) in Bahrainis, Lebanese, and Tunisians was investigated. DRB1*030101-DQB1*0201 was a locus that conferred susceptibility in three populations, while DRB1*040101-DQB1*0302 was a locus that conferred susceptibility only in Tunisians and Bahrainis. The DRB1*100101-DQB1*050101 (Bahrainis) and DRB1*150101-DQB1*060101 (Lebanese) loci were largely protective. The contribution of HLA to T1D must be evaluated with regard to ethnic background.

The relationship between TNF alpha gene polymorphisms (-238/-308), TNF RII VNTR (p75) and outcomes of hepatitis B virus infection in Tunisian population.

The present study was undertaken to investigate the association between Hepatitis B Virus (HBV) infection and polymorphisms of tumour necrosis factor alpha TNF-α -308 G>A, TNF-α -238 G>A and TNF RII VNTR (p75) gene promoter in a Tunisian population. Blood samples were collected from 100 Tunisian patients with HBV infection, 45 with Chronic Hepatitis (CH), 36 with Liver Cirrhosis (LC), 15 with Hepatocellular Carcinoma (HCC) and 200 healthy individuals of similar ethnicity. Genomic DNA was extracted from peripheral blood leukocytes. Genotyping of the analysed polymorphisms was performed using Amplified Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR), Restriction Fragment Length Polymorphism (RFLP) and Variable Number Tandem Repeat PCR (PCR-VNTR). The variant homozygotes -308 GG were associated with 50% decreased risk of HBV chronic infection (GG vs AA+GA; p=0.010; OR=0.50; 95%CI=0.29-0.85). However, the carriers of minor allele -308 A have higher risk (1.5 times) to develop a chronic infection than other patients (p=0.027; OR=1.46; 95%CI=1.04-2.06). The minor allele of -238 polymorphism was positively associated with virus resistance and the development of chronic infection (p=0.043; OR=1.42; 95%CI =1.01 1.99). The distribution of -308, -238 and TNF RII VNTR (p75) among the three groups differed significantly. For HCC groups, there were statistically significant differences in allele distribution in -308, -238 respectively in which A allele remains a risk factor for HBV evolution to HCC (p=0.008 and p=0.026). Haplotype analysis revealed that TNF-α (-308A; -238A) was significantly associated to HBV chronic infection and moreover to disease aggravation to HCC stage. Our findings imply that variations in the genes governing the levels of constitutive and inducible TNF-α and TNF RII might be an important risk factor, which could explain the variable outcomes of HBV infection.

Interleukin-1β, Interleukin1-Ra, Interleukin-10, and tumor necrosis factor-α polymorphisms in Tunisian patients with rheumatoid arthritis.

OBJECTIVES The aim of this study was to investigate the role of IL-1β (-511C>T), TNFα (-308 G>A), IL-10 (-1082 G>A) and IL-1RN VNTR polymorphisms in the susceptibility to rheumatoid arthritis (RA) in Tunisians. PATIENTS AND METHODS Using PCR-based methods, 104 RA patients and 150 healthy controls were investigated. We compared allele and genotype frequencies in RA patients versus controls and analyzed their correlations with erosive form (EF). RESULTS IL1-RN VNTR A1A3 genotype is associated with higher risk of RA (P=0.012, OR=4.31). Among the cases, males who carry this genotype were more exposed to RA (P=0.044, OR=8, 47). For IL1- β gene, a significantly higher frequency of the -511C/C genotype was observed in RA patients in comparison to controls (P=0.013, OR=2.45). This higher frequency was especially observed in women (P=0,003, OR=3.42). In contrast, IL10-1082G/G genotype was less common in patients (P=0.046, OR=0.46). According to EF, men carrying IL1-RN VNTR A1A3 (P=0.005 OR=5.28) and IL1-β-511C/C (P=0.015 OR=2.61) genotypes develop non EF of RA. Moreover, TNFα-308 A allele (P=0.024, OR=1.84) and A/A genotype (P=0.033, OR=3.1) were positively associated to EF of RA. However, G allele (P=0.024, OR=0.31) and GG genotype (P=0.31, OR=0.031) of the TNFα-308 were protectors. CONCLUSION Our results indicated that IL-1RN VNTR, IL-1β (-511C>T) and IL-10 (-1082 G>A) are associated with susceptibility to RA, and that IL-1RN VNTR, IL-1β (-511C>T) and TNFα (-308 G>A) are associated with severity of RA.

Relationship of common vascular endothelial growth factor polymorphisms and haplotypes with the risk of cervical cancer in Tunisians

OBJECTIVE We investigated the association between common vascular endothelial growth factor (VEGF) single nucleotide polymorphisms (SNPs) and the risk of cervical cancer (CC) in Tunisian patients and control women. METHODS Study subjects comprised 86 CC cases and 124 control women. Genotyping of VEGF rs699947, rs833061, rs1570360, rs2010963, rs25648, rs833068, rs833070, rs3025039 SNPs was done by real-time PCR. RESULTS Higher minor allele frequencies (MAF) of rs699947 (-2578C/A) [P=0.04; OR (95% CI)=1.52 (1.02-2.29)], and rs1570360 (-1154G/A) [P=0.04; OR (95% CI)=1.58 (1.01-2.47)] were seen in CC cases compared to control women. Marked differences in the distribution of rs699947 (P=9×10(-4)) and rs1570360 (P=0.03) genotypes were seen between CC cases and control groups; the distribution of the remaining SNPs was comparable between CC cases and control women. The association of rs699947 and rs1570360 with heightened CC risk with was seen in the heterozygous, and more so in the homozygous states. Haploview analysis revealed high LD between rs699947, rs833061, rs1570360, rs2010963, rs25648, rs833068 and rs833070 but weak or no LD between rs3025039 and the other SNPs. Seven-locus (rs699947/rs833061/rs1570360/rs2010963/rs25648/rs833068/ rs833070) haploview analysis identified only CTGCCAG haplotype to be positively associated with CC [P=0.022; OR(95% CI)=1.74 (1.08-2.79)]. CONCLUSION Specific VEGF variants (rs699947, rs1570360) and haplotype (CTGCCAG) may contribute to the development of CC among Tunisian women.

Identification of specific tumor necrosis factor-α-susceptible and -protective haplotypes associated with the risk of type 1 diabetes.

AIM We investigated the association of tumor necrosis factor (TNF)α gene polymorphism with type 1 diabetes (T1D). METHODS TNF-α -1031T/C, -863C/A, -857C/T, -376G/A, -308G/A, -238G/A, and +488G/A single nucleotide polymorphisms (SNPs) were assessed in 198 T1DM patients and 180 age-and gender-matched, normoglycemic control subjects using PCR-restriction fragment length polymorphism (RFLP). RESULTS Higher frequencies of -863A (p = 8.0 × 10-6), -857T (p = 1.4 × 10-4), and -238A (p = 0.002) alleles were seen in T1D patients than in the control group. Significant differences were noted in the distribution of -863T/C, -857C/T, -376G/A, -308G/A, and -238G/A genotypes between patients and controls. Haploview analysis revealed high linkage disequilibrium (LD) between the -376G/A and -308G/A SNPs, but this was lower between the other polymorphisms. Five-locus TNFα haplotypes were constructed based on the prevalence of individual SNPs and the LD between them. An increased frequency of CTGGG, CCGAG, and ACGGG haplotypes, and a reduced frequency of the CCGGG haplotype was seen in patients. When the Bonferroni correction was applied, differences were significant for the CTGGG (Pc = 1.4 × 10-3), CCGAG (Pc = 0.023), and ACGGG (Pc = 1.2 × 10-3) haplotypes which were greater, and the CCGGG haplotype (Pc = 3.8 × 10-5) which was smaller, among T1D patients, thereby conferring susceptibility to and protection from T1D, respectively. CONCLUSION These results demonstrate that TNF-α polymorphisms, in particular -863C/A, -857C/T, and -238G/A, are significantly associated with T1D. Additional studies, on other racial groups, are needed to confirm our findings.

Glutamic Acid Decarboxylase 65 and Islet Cell Antigen 512/IA-2 Autoantibodies in Relation to Human Leukocyte Antigen Class II DR and DQ Alleles and Haplotypes in Type 1 Diabetes Mellitus

ABSTRACT The frequencies of autoantibodies against glutamic acid decarboxylase 65 (GAD65) and islet cell antigen (ICA) 512/IA-2 (512/IA-2) are functions of the specific human leukocyte antigen (HLA) in type 1 diabetes mellitus (T1D). We investigated the association of HLA class II (DR and DQ) alleles and haplotypes with the presence of GAD and IA-2 autoantibodies in T1D. Autoantibodies were tested in 88 Tunisian T1D patients and 112 age- and gender-matched normoglycemic control subjects by enzyme immunoassay. Among T1D patients, mean anti-GAD antibody titers were higher in the DRB1*030101 allele (P < 0.001), together with the DRB1*030101/DQB1*0201 (P < 0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes, while lower anti-GAD titers were associated with the DRB1*070101 (P = 0.001) and DRB1*110101 (P < 0.001) alleles and DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1*030101 (P = 0.001) haplotypes. Mean anti-IA-2 antibody titers were higher in the DRB1*040101 allele (P = 0.007) and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes but were lower in the DRB1*110101 allele (P = 0.010) and the DRB1*110101 (P < 0.001) and DRB1*110101/DQB1*030101 (P = 0.025) haplotypes. Multinomial regression analysis confirmed the positive association of DRB1*030101 and the negative association of DRB1*110101 and DQB1*030101, along with the DRB1*070101/DQB1*0201 and DRB1*110101/DQB1*030101 haplotypes, with anti-GAD levels. In contrast, only the DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-IA-2 titers. Increased GAD65 and IA-2 antibody positivity is differentially associated with select HLA class II alleles and haplotypes, confirming the heterogeneous nature of T1D.

IL-10 gene promoter and intron polymorphisms as genetic biomarkers of cervical cancer susceptibility among Tunisians

OBJECTIVE We investigated the association between polymorphisms in the promoter and intron regions of the interleukin-10 (IL-10) gene with the risk of cervical cancer (CC) in Tunisian patients and control women. METHODS Study subjects comprised 86 CC cases and 126 control women. Genotyping of IL-10 intron (rs3024491, rs3024490) and promoter (rs1800872, rs1800871, rs1800896) variants was done by real-time PCR, with defined clusters. RESULTS The minor allele frequencies of the five tested IL-10 SNPs were not significantly different between cervical cancer cases and control women. However, significantly higher frequencies of homozygous minor allele-carriers in cases was seen for rs3024490 (P=0.023), rs1800872 (P=0.037), and rs1800871 (P=0.028). IL-10 serum levels were significantly reduced in rs3024490 T/T vs. G/G genotype carriers, and in rs1800871 T/T than C/C genotype carriers. While carriage of rs1800872 and rs3024491 minor allele was associated with reduced IL-10 secretion, this was not statistically significant. Haploview analysis demonstrated high linkage disequilibrium (LD) among the IL10 SNPs studied, and only seven haplotypes were common, capturing 98.8% of the total possible haplotypes. Reduced frequency of haplotypes GTCCA (P<0.001) and TGATG (P<0.001) was seen in cervical cancer cases than in control women, thus conferring disease protection nature to these haplotype. This association remained significant for GTCCA (Pc=0.006) and TGATG (P=0.045) after correcting for multiple comparisons. CONCLUSION Specific IL-10 variants (rs3024490, rs1800872, and rs1800871) and haplotype (GTCCA and TGATG) may contribute to the development of cervical cancer among Tunisian women.

Modulation of Type 1 Diabetes Susceptibility by Tumor Necrosis Factor Alpha −308 G/A and Lymphotoxin Alpha +249 A/G Haplotypes and Lack of Linkage Disequilibrium with Predisposing DQB1-DRB1 Haplotypes in Bahraini Patients

ABSTRACT Tumor necrosis factor alpha (TNF-α) −308 G/A and lymphotoxin alpha (LTα) +249 A/G single-nucleotide polymorphisms were investigated in 228 type 1 diabetes mellitus (T1DM) patients and 240 controls. Only LTα +249G allele and +249G/+249G genotype frequencies were higher among patients, and no linkage disequilibrium was found between TNF-α/LTα alleles and susceptible/protective DRB1-DQB1 haplotypes. TNF-α/LTα T1DM-susceptible (−308G/+249G) and protective (−308G/+249A) haplotypes were identified.

Association of BANK1 and cytokine gene polymorphisms with type 1 diabetes in Tunisia.

Type 1 diabetes (T1D) is an autoimmune disease (AID) with both genetic and environmental components. We aimed to investigate the genetic association of polymorphisms in genes previously linked with other AIDs, namely BANK1, IL15 and IL2/IL21 region. A total of 76 T1D patients and 162 controls from Southern Tunisia were recruited for a case-control association study investigating the relationship between sixteen SNPs of the BANK1, IL15 and IL2/IL21 gene region and T1D. In the BANK1 gene, G allele and GG genotype of rs3733197 were significantly increased in the group of T1D patients compared to controls. In addition, in the IL15 gene, the minor allele A of rs10519613 polymorphism was significantly higher in patients than in controls. No significant association was found for SNPS in IL2/IL21 gene region. The analysis of the haplotype structure revealed the G-C-A-C-T haplotype of the IL15 gene as associated with a reduction in the risk of developing T1D, while A-T-A-C-T haplotype increased the risk of developing the disease. Furthermore, in the IL2/IL21 region, only one haplotype consisting of eight SNPs was markedly associated with T1D susceptibility. Moreover, G-C combination of the BANK1/IL15 was significantly increased in T1D patients, compared to controls. Our results establish BANK1 and IL15 as new T1D genetic susceptibility factors and replicate the association of the 4q27 region with T1D. Our data agree with the effect previously observed for other autoimmune conditions and delineate a shared underlying mechanism.